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Abstract Background Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. Graphical abstract

Abstract A pattern of increasing species richness from the poles to the equator is frequently observed in many animal taxa. Ecological limits, determined by the abiotic conditions and biotic interactions within an environment, are one of the major factors influencing the geographical distribution of species diversity. Energy availability is often considered a crucial limiting factor, with temperature and productivity serving as empirical measures. However, these measures may not fully explain the observed species richness, particularly in marine ecosystems. Here, through a global comparative approach and standardised methodologies, such as Autonomous Reef Monitoring Structures (ARMS) and DNA metabarcoding, we show that the seasonality of primary production explains sessile animal richness comparatively or better than surface temperature or primary productivity alone. A Hierarchical Generalised Additive Model (HGAM) is validated, after a model selection procedure, and the prediction error is compared, following a cross-validation approach, with HGAMs including environmental variables commonly used to explain animal richness. Moreover, the linear effect of production magnitude on species richness becomes apparent only when considered jointly with seasonality, and, by identifying world coastal areas characterized by extreme values of both, we postulate that this effect may result in a positive relationship in environments with lower seasonality.

Abstract The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021–2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.

Amphibians are among the most threatened vertebrates globally, and their conservation status continues to decline. In the updated Global Amphibian Assessment (GAA2), the use of IUCN Criterion E, which projects extinction risks through quantitative models, highlighted southern Europe as a hotspot for salamander extinction risk due to the risk of invasion by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). In particular, for five Italian salamander species, risk categories were elevated significantly based on Criterion E, from Vulnerable or lower to Endangered or Critically Endangered. This increased reliance on Criterion E raises concerns regarding its treatment of uncertainty, as these projections depend heavily on assumptions about Bsal spread, environmental suitability, and host dynamics. Limited exploration of alternative scenarios and reliance on extreme parameter values ​​may result in inflated extinction risk estimates. We emphasize the need for improved documentation of uncertainty and integration of diverse expert opinions in extinction risk assessments, to balance proactive conservation planning with robust scientific methodology.

Summary: Interspecific hybridization can be consequential for insular species. The Critically Endangered Aeolian wall lizard, Podarcis raffonei, severely declined due to interactions with the invasive Italian wall lizard, Podarcis siculus. The largest population of P. raffonei survives on a narrow peninsula (Capo Grosso) that is mildly connected to the island of Vulcano, which has been entirely invaded by P. siculus. Recent observation of individuals with an intermediate phenotype raised concern over the risk that hybridization might swamp this last stronghold. We genetically characterized lizards from Vulcano, considering individuals showing multiple phenotypes (native, invasive, and “intermediate”). Hybridization rate was low (∼3%), with just two F1 hybrids and two backcrosses, suggesting that hybridization does not currently represent a major threat. However, we identified low genetic diversity, a small effective population size, and a low Ne/Nc ratio. Management strategies are urgently needed to control invasive species and maintain the genetic diversity of P. raffonei.

Competition between native and alien species is often described as a main driver of biodiversity loss. Nevertheless, there is limited evidence of animal declines and extinctions actually determined by competition. The Aeolian lizard, Podarcis raffonei, is critically endangered because it suffered dramatic declines and extinctions throughout its range. Competition and hybridization with invasive Italian lizards, Podarcis siculus, have been proposed as a driver of the shrinkage of Aeolian lizards, still the mechanisms underlying their decline remain poorly resolved. We used observations of behavioral encounters, combined with morphological data and robust species identification based on genomics, to test whether agonistic interactions with the invasive Italian lizard can explain the competitive exclusion and rapid decline of the native Aeolian lizard while accounting for hybridization. Invasive lizards were larger, with larger heads, and showed higher bite tendency against neutral items. In agonistic encounters between males, Aeolian lizards received more attacks and escaped more frequently than invasive males. The performance of Aeolian males was particularly poor in interspecific encounters. Genomic data verified that tested individuals were all pure P. raffonei or P. siculus, with a single hybrid individual detected. The strong competitive advantage of invasive males can allow them monopolizing territories, potentially hampering the reproduction of both native males and females, thus resulting in a mechanism of sterilizing interference. Reproductive interference competition mediated by spatial exclusion might be an unappreciated process determining rapid decline in endemic species. Safeguarding areas devoid of invasive species should be the priority strategy to avoid the extinction of the Aeolian lizards.

Abstract The new key biodiversity areas (KBA) standard is an important method for identifying regions of the planet‐hosting unique biodiversity. KBAs are identified through the implementation of threshold‐based criteria that can be applied to any target species and region. Current methods to rapidly assess the existence of potential KBAs in different areas of the planet still present important challenges, although they are needed to accelerate the KBA identification process for large numbers of species globally. We developed a methodology to scan geographical regions and detect potential KBAs under multiple criteria. We tested the methodology on 59 species of reptiles and amphibians in Italy through the application of selected KBA criteria. Potential KBAs were identified for multiple species under most criteria, covering 1.4%–12% of the study area, depending on analytical settings. Cell size used to identify KBAs played an important role in shaping the distribution of potential KBAs, also affecting the overlap between areas triggered by different criteria. New potential KBAs identified in this study are only partially coincident with current KBAs in Italy (previously identified for birds) and within the national protected areas.

Individual diet specialization is a key behavioral trait, with multifaceted impacts on population dynamics. Long-term studies are fundamental to properly understand such dynamics and their main causes. Indeed, individuals may vary from specialism to generalism in response to multiple biological and environmental factors, thus snapshot studies can have limited relevance for the overall biology of species. Yet, so far longitudinal studies testing the potential variation of diet specialization through time are lacking. Here we present the results of a study on individual diet specialization performed over a four-year period. Using three populations of the Ambrosis’ cave salamander Speleomantes ambrosii we tested: i) whether seasonal variation in the occurrence of specialized individuals is maintained through different years, and ii) if the degree of specialization observed in a single season is constant through different years. Our results showed that the species maintains a high proportion of specialized individuals in spring and more generalist in autumn during different years, confirming a consistent seasonal variation of trophic specialization through time. On the other hand, the proportion of specialized individuals of the same period can vary up to ten-fold across years, suggesting that a single study may be not enough to properly understand the dynamics of a species’ trophic niche. This study highlights the need of repeated surveys to accurately understand this behavioral trait, especially for species that are strongly dependent on local environmental conditions.

Abstract Landscapes nearby glaciers are disproportionally affected by climate change, but we lack detailed information on microclimate variations that can modulate the impacts of global warming on proglacial ecosystems and their biodiversity. Here, we use near-subsurface soil temperatures in 175 stations from polar, equatorial and alpine glacier forelands to generate high-resolution temperature reconstructions, assess spatial variability in microclimate change from 2001 to 2020, and estimate whether microclimate heterogeneity might buffer the severity of warming trends. Temporal changes in microclimate are tightly linked to broad-scale conditions, but the rate of local warming shows great spatial heterogeneity, with faster warming nearby glaciers and during the warm season, and an extension of the snow-free season. Still, most of the fine-scale spatial variability of microclimate is one-to-ten times larger than the temporal change experienced during the past 20 years, indicating the potential for microclimate to buffer climate change, possibly allowing organisms to withstand, at least temporarily, the effects of warming.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.

Summary: Emojis enable direct expressions of ideas and emotions in digital communication, also contributing to discussions on biodiversity conservation. Nevertheless, the ability of emojis to represent the Earth’s tree of life remains unexplored. Here, we quantified the taxonomic comprehensiveness of currently available nature-related emojis and tested whether the expanding availability of emojis enables a better coverage of extant biodiversity. Currently available emojis encompass a broad range of animal species, while plants, fungi, and microorganisms are underrepresented. Within animals, vertebrates are significantly overrepresented compared to their actual richness, while arthropods are underrepresented. Notwithstanding these taxonomic disparities, animal taxa represented by emojis more than doubled from 2015 to 2022, allowing an improved representation of both taxonomic and phylogenetic diversity, driven by the recent addition of cnidarians and annelids. Creating an inclusive emoji set is essential to ensure a fair representation of biodiversity in digital communication and showcase its importance for biosphere functioning.

Abstract Invasion dynamics are determined, among other aspects, by the spatial behaviour of invasive populations. The invasive toad Duttaphrynus melanostictus is spreading inland from the eastern coast of Madagascar, causing considerable ecological impacts. Understanding the basic factors determining the spread dynamics can inform management strategies and provide insights into spatial evolutionary processes. We radio-tracked 91 adult toads in three localities along the invasion gradient to determine whether spatial sorting of dispersive phenotypes is occurring, and investigate intrinsic and extrinsic determinants of spatial behaviour. Overall, toads in our study appeared as habitat generalists, and their sheltering behaviour was tied to water proximity, with toads changing shelter more frequently closer to waterbodies. Toads showed low displacement rates (mean = 4.12 m/day) and quite a philopatric behaviour but were able to perform daily movements of over 50 m. We did not detect any spatial sorting of dispersal-relevant traits nor sex- or size-biased dispersal. Our results suggest that toads are more likely to expand their range during the wet season, and that the range expansion is probably dominated by short-distance dispersal at this stage of the invasion, although a future increase in invasion speed is expected, due to the capacity for long-distance movements of this species.

Abstract Experimental introductions of species have provided some of the most tractable examples of rapid phenotypic changes, which may reflect plasticity, the impact of stochastic processes, or the action of natural selection. Yet to date, very few studies have investigated the neutral and potentially adaptive genetic impacts of experimental introductions. We dissect the role of these processes in shaping the population differentiation of wall lizards in three Croatian islands (Sušac, Pod Kopište, and Pod Mrčaru), including the islet of Pod Mrčaru, where experimentally introduced lizards underwent rapid (~30 generations) phenotypic changes associated with a shift from an insectivorous to a plant‐based diet. Using a genomic approach (~82,000 ddRAD loci), we confirmed a founder effect during introduction and very low neutral genetic differentiation between the introduced population and its source. However, genetic depletion did not prevent rapid population growth, as the introduced lizards exhibited population genetic signals of expansion and are known to have reached a high density. Our genome‐scan analysis identified just a handful of loci showing large allelic shifts between ecologically divergent populations. This low overall signal of selection suggests that the extreme phenotypic differences observed among populations are determined by a small number of large‐effect loci and/or that phenotypic plasticity plays a major role in phenotypic changes. Nonetheless, functional annotation of the outlier loci revealed some candidate genes relevant to diet‐induced adaptation, in agreement with the hypothesis of directional selection. Our study provides important insights on the evolutionary potential of bottlenecked populations in response to new selective pressures on short ecological timescales.

Abstract The problem of global amphibian declines has prompted extensive research over the last three decades. Initially, the focus was on identifying and characterizing the extent of the problem, but more recently efforts have shifted to evidence‐based research designed to identify best solutions and to improve conservation outcomes. Despite extensive accumulation of knowledge on amphibian declines, there remain knowledge gaps and disconnects between science and action that hamper our ability to advance conservation efforts. Using input from participants at the ninth World Congress of Herpetology, a U.S. Geological Survey Powell Center symposium, amphibian on‐line forums for discussion, the International Union for Conservation of Nature Assisted Reproductive Technologies and Gamete Biobanking group, and respondents to a survey, we developed a list of 25 priority research questions for amphibian conservation at this stage of the Anthropocene. We identified amphibian conservation research priorities while accounting for expected tradeoffs in geographic scope, costs, and the taxonomic breadth of research needs. We aimed to solicit views from individuals rather than organizations while acknowledging inequities in participation. Emerging research priorities (i.e., those under‐represented in recently published amphibian conservation literature) were identified, and included the effects of climate change, community‐level (rather than single species‐level) drivers of declines, methodological improvements for research and monitoring, genomics, and effects of land‐use change. Improved inclusion of under‐represented members of the amphibian conservation community was also identified as a priority. These research needs represent critical knowledge gaps for amphibian conservation although filling these gaps may not be necessary for many conservation actions.

Abstract The determination of a species trophic niche can clarify its functional role within a food web and how prey resources are used in relation with the spatial and temporal variability of environmental conditions. This information may result particularly useful for the implementation of conservation plans of endangered species having a cryptic behaviour or living in places difficult to be surveyed. Here we present the first long-term study on the trophic niche of the Italian cave salamander Speleomantes italicus, a strictly protected facultative cave species that seasonally exploits surface environments (e.g., forested areas) as well as both natural and artificial subterranean environments. We analysed the diet variation of six populations of S. italicus inhabiting natural caves, surveyed 24 times in a full year. During the surveys, all sampled individuals were subjected to stomach flushing and the ingested prey were identified and enumerated; furthermore, salamanders’ body condition was also evaluated. The results of the analyses provided the first comprehensive, year-round assessment of the diet for a Speleomantes species. Remarkable divergences in terms of trophic niche and body condition were observed between the studied populations. We found a discrepancy in the foraging activity of the populations located in different areas; specifically, the individuals that experienced sub-optimal microclimatic conditions poorly performed in foraging. Furthermore, we found temporal and spatial variability in the body condition of individuals. Our study highlighted a remarkably high spatial and temporal divergence in the trophic habits of conspecific populations, a feature that may represent one of the major factors promoting the variability of multiple population traits.

Abstract The evolution of sexual dimorphism (SD) is driven by intricate interplays between sexual and natural selection. When it comes to SD variation within populations, however, environmental factors play a major role. Sexually selected traits are expected to be strongly dependent on individual body condition, which is influenced by the local environment that individuals experience. As a consequence, the degree of SD may also depend on resource availability. Here, we investigated the potential drivers of SD expression at two sexually dimorphic morphometric traits, body size (snout vent length) and head shape (head geometric morphometrics), in the Italian wall lizard (Podarcis siculus). We assessed the existence of condition- and context-dependent SD across ten islands of the Aeolian archipelago (southern Italy), at within- and among-population scales. We observed strong geographical variation of SD among islands, and tested three potential SD predictors related to resource availability (individual body condition, ecosystem productivity, temperature). Body condition and ecosystem productivity were the main drivers of body size SD variation, and body condition was also the main driver for head shape SD. Our results highlight that the expression of SD in the Italian wall lizard is both condition- and context-dependent. These results are congruent at within- and among-populations scales highlighting that spatial multi-scale analysis represents a useful approach to understand patterns of SD expression.

Abstract Aposematic bright colors have a key role for animal defense and can be expressed through metabolic production or by acquiring pigments from diet. Aposematic coloration can be related to both local adaptations and availability of trophic resources. The European fire salamander (Salamandra salamandra) shows significant color variability and occurs across a broad range of habitats. Here we combined field observations with common rearing experiments to disentangle the role of environmental conditions and local adaptations in determining aposematic coloration of salamander populations. We assessed color variation and measured habitat features and food availability in adults from 25 populations. Furthermore, we reared newborn larvae from 10 populations under different food availability and analyzed color of metamorphs. To assess color pattern, we measured the percentage of yellow covering the body, and the Hue, Saturation and Value of yellow coloration. Adult showed strong variation of color pattern; variation was strongly related to the individual's size, to habitat productivity and to food availability. Under common garden conditions, differences between populations were not anymore evident, and coloration was only affected by resource availability during larval development. Our results suggest that environmental conditions and food availability are more important than local adaptations in determining differences in aposematic color pattern.

Planning conservation actions requires detailed information on species’ geographic distribution. Species distribution data are most needed in areas hosting unique or endangered biodiversity. Italy is one of the European countries with the highest levels of herpetological diversity and endemism and is home to several threatened species of amphibians and reptiles. Information on the distribution of species’ habitats can help identify sites where the species is most likely to thrive, as viable populations depend on it. Area of Habitat (AOH) maps reveal the distribution of the habitat available to the species within their geographic range. We produced high resolution, freely accessible global area of habitat maps for 60 species of reptiles and amphibians distributed in Italy, which represent 60% of all Italian amphibian and reptile species. We validated a total of 44 AOH maps through a presence-only based evaluation method, with 86% of these maps showing a performance better than expected by chance. AOH maps can be used as a reference for conservation planning, as well as to investigate macroecological patterns of Italian herpetofauna. Furthermore, AOH maps can help monitoring habitat loss, which is known to be a major threat to many reptile and amphibian species in Europe.

Invasive predators can strongly affect native populations. If alien predator pressure is strong enough, it can induce anti-predator responses, including phenotypic plasticity of exposed individuals and local adaptations of impacted populations. Furthermore, maternal investment is an additional pathway that could provide resources and improve performance in the presence of alien predators. We investigated the potential responses to an alien predator crayfish (Procambarus clarkii) in a threatened frog (Rana latastei) by combining field observations with laboratory measurements of embryo development rate, to assess the importance of parental investment, origin and exposure to the crayfish cues. We detected a strong variation in parental investment amongst frog populations, but this variation was not related to the invasion status of the site of origin, suggesting that mothers did not modulate parental investment in relation to the presence of alien predators. However, cues of the invasive crayfish elicited plastic responses in clutches and tadpoles development: embryos developed faster when exposed to the predator. Furthermore, embryos from invaded sites reached Gosner’s development stage 25 faster than those from non-invaded sites. This ontogenetic shift can be interpreted as a local adaptation to the alien predator and suggests that frogs are able to recognise the predatory risk. If these plastic responses and local adaptation are effective escape strategies against the invasive predator, they may improve the persistence of native frog populations.

The salamander killing fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia, is threatening salamander populations in different parts of Europe. In fact, this pathogen is rapidly spreading in Central Europe and has been also introduced into NE Spain. Of special concern are those regions with an exceptionally high salamander diversity such as Italy, where 19 salamander species are present most of which are strictly endemic. In this study, we update the information on the presence of Bsal in Italian wild salamanders, by adding samples from two presumptive outbreak sites, one on the island of Sardinia and one on continental Italy (Liguria). In addition, we reviewed the potential susceptibility of all the Italian salamander species on the basis of laboratory experimental infection trials, or from the tested susceptibility of the phylogenetically nearest species, according to the literature. Overall, 15 skin swabs from three species (Speleomantes sarrabusensis, Speleomantes strinatii and Salamandra salamandra) collected in the two presumptive Bsal outbreak sites were analysed by quantitative molecular methods, but none gave positive results. The majority of Italian salamander species and almost all of the endemic ones showed a high susceptibility to Bsal infection. Therefore, even if the presence of Bsal in Italian salamander populations has not been proven yet, the entire salamander fauna is highly threatened and preventive management actions should be taken. The need for strict biosecurity protocols on the international trade of captive or wild amphibians and for the implementation of preventive measures during field activities to limit the introduction and spread of the Bsal pathogen is again stressed.

Detecting the trends of species and populations is fundamental to identifying taxa with high conservation priority. Unfortunately, long-term monitoring programs are challenging and often lacking. The Italian agile frog Rana latastei is endemic to Northern Italy and adjacent countries, is considered vulnerable by the IUCN, and is protected at the European level. However, quantitative estimates of its decline are extremely scarce. In this study, we document the trends in abundance and distribution of Rana latastei within Monza Park, which currently represents the area closer to the type locality of the species and holds unique genetic features. Wetlands within the park were monitored from 2000 to 2023; counts of egg clutches were taken as a measure of reproductive output and the abundance of breeding females. In 2000, the species occurred over a significant proportion of the park. Total abundance showed strong yearly variation but remained rather constant from 2000 to 2019. However, Rana latastei disappeared from the park around 2021 and was never detected in 2022–2023. The decline is probably related to the joint effect of multiple factors, including the conversion of breeding sites for farming, inappropriate water management, invasive alien species, and severe drought. The local extinction of Rana latastei occurred despite legal protection, highlighting the need for more effective and stringent tools for the conservation of European biodiversity.

Measurement(s) Gastric Content • Image Technology Type(s) light microscopy • Digital Photography Sample Characteristic - Organism Speleomantes • Hydromantes Sample Characteristic - Environment Subterranean • karst cave • mine Sample Characteristic - Location Italy • Region of Liguria • Municipality of Genova • Province of La Spezia Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14346176

Abstract Assessing biodiversity loss and species extinction is necessary to warn society and raise awareness of the impacts of ongoing climate change. Prioritizing protected areas is the pragmatic and applicable management measure under the pressure of ongoing climate change and limited resources to conserve species at risk of extinction. We developed a novel conservation index (CI) to prioritize areas and populations of an endangered mountain tree species that need protection in the face of ongoing climate change, as conservation of all populations may not be realistic. This CI integrates (1) mountain topography to identify potential refugial areas with suitable microclimates, (2) genetic diversity to assess the adaptive capacity of local populations, and (3) hypothetical climate change in the species' range. We applied this CI to Atlas cedar, an endemic and threatened species whose populations are scattered throughout the Moroccan mountains. This index provided a scale for 33 populations studied and suggests that genetically diverse populations located in rugged areas where future local climate may overlap with their current climatic niche should receive a higher conservation priority. This index may also be applicable to other mountain species with scattered populations and is likely to be more accurate if more precise climate data are used at the microrefugia scale.

Abstract The worldwide biodiversity crisis with the resulting need to increase species protection has led researchers to pursue and select survey methods that guarantee the best quality of data and produce the least negative effects on wild animals. Plethodontids are the most diverse family of salamanders; all species are very sensitive to human handling and noninvasive, but accurate, measurement methods are needed to reduce researchers’ impact. Here, we tested the reliability of a noninvasive post hoc method in estimating the snout–vent length (SVL) from photographs showing salamanders’ dorsal view. The correlation between the estimated snout–vent length (SVLe) and the conventional SVL was high (R2m = .81), and no significant difference occurred between operators with different experience. Finally, we list the numerous advantages for the use of SVLe in terms of data quality and in reducing the stress caused to wild animals.

The European Union (EU) has recently adopted a regulation on invasive alien species that foresees the possibility of developing lists of species of National Concern. We developed a prioritisation process for alien mammals already established in Italy, but not yet included in the EU list (n = 6 species) and a systematic horizon-scanning procedure to obtain ranked lists for those species that are already introduced worldwide or traded in Italy (n = 213). Experts were asked to score these species, by evaluating their likelihood of establishment and spread and the magnitude of their potential impacts on biodiversity, economy, human-health and society. The manageability of each species was also evaluated, both for the proritisation and the horizon-scanning processes. We produced five lists that ranked species according to their potential spread and impacts and their manageability. These will allow policy-makers to select outputs according to a balance between risk assessment and risk management, establishing priorities for alien species management at the national level.

Invasive species can influence the evolution of natives. Here, authors use common garden experiments to show that invasive predatory crayfish have homogenized the developmental timing of a native frog by selecting for more rapid development to avoid predation.

Abstract Sedimentary ancient DNA (SedaDNA) is an emerging tool to reconstruct past biodiversity with high taxonomic resolution. Its growing popularity has stimulated an increasing complexity of SedaDNA data production (e.g., DNA extraction, amplification, and sequencing; authentication of molecules; bioinformatics). Conversely, less attention has been devoted to how appropriate statistical analyses can help to extract ecological information from SedaDNA. Until now, ecological studies based on SedaDNA have taken limited advantage of the multiple statistical and numerical methods available for analysis. Here, we present a range of numerical approaches that can be particularly useful to multispecies ecological analysis on SedaDNA, with a special focus on biodiversity studies on macroorganisms. We discuss the advantages and complexity of such methods and describe how some of them can be optimized for ecological analyses of SedaDNA‐based metabarcoding data, with a special focus on SedaDNA studies. First, site occupancy‐detection models can help to better ascertain the variation through time of the occurrence of target species and to identify the factors determining their detection through time. Second, several approaches can be used to estimate variation of relative abundance. Even though methods for abundance estimation have major limitations, they can provide useful information on temporal variation of ecosystem functions. Third, approaches exist to obtain better measures of species diversity, while taking into account the uncertainties of species abundance and identification. Fourth, techniques of clustering, ordination, and constrained ordination allow identification of temporal trends and testing of candidate drivers of community variation. Finally, structural equation models can be used to assess complex causal relationships among biodiversity, human activities, and environment. SedaDNA studies can make use of a broad panel of analytical approaches, which can improve our understanding of long‐term biodiversity changes, maximizing the information we can obtain from past ecosystems.

We describe and delimit with integrative taxonomy the new springtail species Desoria calderonis sp. nov. (Collembola: Isotomidae). This cryophilic species is strictly linked to the supraglacial stony debris of the isolated Calderone glacier (Central Apennines, Italy), one of the southernmost glaciers of Europe. Desoria calderonis sp. nov. could belong to the nivalis-complex, a group of European mountain species included in the violacea-group. Genetic analysis (COI mtDNA barcoding) confirms the morphological attribution to the genus Desoria Nicolet in Desor, 1841, but highlights that the genus, in its current definition, is polyphyletic. We specify the peculiar micro-habitat preferences and highlight the threat of extinction for this cryophilic species in the context of the ongoing climate change and subsequent risk of complete disappearance of the glacier.

Photographic identification is an emerging method for recognising wild animals. This harmless methodology allows researchers to identify “naturally marked” individuals and therefore study their specific ecology and behaviour. However, before incurring potential data loss, it is recommended to test the methodology on the target species and evaluate the pros and cons. We assessed the reliability of photographic identification in adult Hydromantes salamanders from three species. Specifically, we assessed whether the dorsal pattern of adult salamanders changed over time, thus evaluating its potential use as a reliable marking methodology. We used capture-mark-recapture and controlled conditions (i.e. individuals kept in fauna boxes) to evaluate potential changes in the dorsal pattern of Hydromantes through time. We did not observe any change in the dorsal pattern in the three species during the study period. Photographic identification might be a useful marking technique for these endangered species. However, these animals are usually found in environments generally lacking light and thus, researchers must be careful in setting up proper light conditions to produce suitable pictures for individual identification of Hydromantes.

In Europe, invasive freshwater crayfish are not only changing freshwater ecosystems, but they are also leading to local extinctions of native freshwater crayfish. This is particularly evident for the populations of red swamp crayfish and spiny-cheek crayfish in northern Italy, which are threatening the last and isolated populations of the white-clawed crayfish. Here, we describe the steps that accompanied a successful reintroduction of the white-clawed crayfish in an Italian stream (Park Monte Barro) that, although isolated from other freshwater sites, suffered from an illegal introduction of the spiny-cheek crayfish in 2013. After the removal of presumably all the introduced spiny-cheek crayfish individuals, we started periodical surveys (twice a year) of the stream to confirm the absence of further introductions and to monitor environmental conditions. Prior to the reintroduction of the white-clawed crayfish that started in autumn 2018, we developed an intense dissemination activity to raise awareness of white-clawed crayfish features and importance among landowners surrounding the stream, including those suspected of the introduction of the spiny-cheek crayfish: we organized public meetings and we performed seven direct visits, house to house, to the local people providing information on good practices for white-clawed crayfish conservation. From 2018 to 2020, every autumn, we reintroduced a batch of 3-month-old white-clawed crayfish juveniles, and we developed a program for the monitoring of crayfish growth and density, water quality, and direct landowners’ disturbance of the site. We detected a significant increase of the white-clawed crayfish total length (TL) from the first reintroduction (October 2018) to June 2020. In 2020, crayfish were consistently larger than in the 2019 surveys; some of them were able to breed less than 2 years after the first reintroduction. In 2020, the estimated density of large crayfish reached 0.57 individuals/m2, which is lower than the density observed prior to extinction. We did not detect any case of human disturbance of the site. Our results underline that the reintroduction actions could be more effective when the stakeholders having the greatest potential impact on the species are identified, informed, and involved as primary caretakers of the activities.

Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed.

Reduced trophic resources can pose relevant constraints to the development of freshwater animals with complex life cycles. For amphibians, food deprived environments, such as high-altitude ponds and springs and groundwaters are frequently used for breeding. The aim of this study is to outline trophic conditions leading to extreme cases of delayed larval development and increased size at metamorphosis of a European widespread amphibian, the fire salamander (Salamandra salamandra). We collected 150 fire salamander larvae, split them in two groups, one with high and one with low trophic resource availability. We then observed the effects of nutritional conditions on larval development recording time to metamorphosis and average day growth. Moreover, in the field, we surveyed larvae growth and size at metamorphosis in two artificial subterranean sites with low prey availability. Trophic conditions strongly affected larval development and under low food treatment time to metamorphosis reached up to 416 days. In the subterranean environments we observed a similar pattern, with larvae requiring more than one year to attain metamorphosis but reaching unexpected large sizes. Environmental trophic conditions experienced during early stages can induce strong delay in metamorphosis of the fire salamander; this plasticity makes fire salamander larvae optimal models for comparative studies and cross-environment experiments.

The Aeolian wall lizard Podarcis raffonei is an island endemic that survives only on three tiny islets, and on the Capo Grosso peninsula of the Vulcano island, thus is among the European vertebrates with the smallest range and one of the most threatened by extinction. This species is declining due to competition and hybridization with the non-native lizard Podarcis siculus, but a regular monitoring program is lacking. Here we assessed the size and status of the Capo Grosso population of P. raffonei on Vulcano. In September 2015 we captured 30 individuals showing the typical brown phenotype of P. raffonei, while one single male showed a green phenotype, apparently intermediate between P. raffonei and the non-native Podarcis siculus. In May 2017, only 47% of 131 individuals showed the typical brown phenotype (P. raffonei-like) and 53% showed the green phenotype (P. siculus-like). Based on N-mixture models and removal sampling the estimated size of the Capo Grosso population was of 800-1300 individuals in 2017, being similar to 2015; available data suggest that the total range of the species could be as small as 2 ha. The frequency of individuals with the typical P. raffonei phenotype dramatically dropped between two samplings with a parallel increase of individuals displaying the green phenotype. Observation on outdoor captive-bred individuals demonstrates plasticity for colouration in P. raffonei individuals from Capo Grosso, with several individuals showing the typical brown pattern in September 2017 and a green pattern in March 2021. Non-exclusive hypotheses, including hybridization with P. siculus and plasticity in colour pattern of P. raffonei, are discussed to explain the phenotypic shifts of the P. raffonei population of Capo Grosso. While genomic evidence is required to reach conclusions and investigate eventual hybridization, it is urgent to undertake a programme for the monitoring and management of this lizard.

Recently described species suffer lack of information that hampers setting up appropriate conservation strategies. The situation is particularly complex with micro-endemic snakes, for which detection and monitoring are particularly challenging. The Walser viper Vipera walser is a recently described snake inhabiting a small area of the SW Italian alps. We combined information on species distribution with repeated monitoring to identify the areas most suitable for the species, and to obtain estimates of species abundance. Species distribution models were used to identify the topographical, climatic, and land-cover features related to the occurrence of vipers. Furthermore, repeated transects and N-mixture models were used to estimate abundance and to identify factors related to the variation of abundance. The available data suggested that the species has a disjunct range, with a Northern range of ~45 km2, and a southern range of ~225 km2. Distribution models suggested that vipers are associated with areas with open vegetation, altitude between 1300 and 2300 m, high precipitation, low forest cover, low slope, and southern aspect. N-mixture models confirmed very low detection probability of these vipers, and suggested that the species has a low abundance, with the highest abundance in south-facing plots. We provide the first quantitative information on habitats and abundance variation for Walser vipers. The broad confidence intervals of abundance estimates exemplify the complexity of providing range-wide measures of abundance for secretive species. Given the narrow range of these vipers, continuous monitoring is required to understand how they respond to ongoing environmental changes in mountainous areas.

Microhabitat selection is a key component of amphibian breeding biology and can be modulated in response to the features of breeding sites and the presence of predators. Despite invasive alien species being among the major threats to amphibians, there is limited information on the role of invasive species in shaping amphibians’ breeding microhabitat choice. The invasive red swamp crayfish (Procambarus clarkii) is a major predator of amphibians’ larvae, including those of the brown frogs Rana dalmatina and Rana latastei. Although qualitative information about the spawning site preferences and breeding microhabitat choice of brown frogs is available in the literature, only a few studies performed quantitative analyses, and the relationship between microhabitat choice and the presence of alien predators has not been investigated yet. The aims of this study were: (1) to characterize the microhabitats selected for clutch deposition by R. dalmatina and R. latastei and (2) to test if the position and the aggregation of egg clutches differ in sites invaded or not invaded by P. clarkii. During spring 2017, we surveyed multiple times 15 breeding sites of both brown frogs in Northern Italy; in each site we assessed the features of the microhabitat where each egg clutch was laid, considering its position (distance from the shore, depth of the water column) and the degree of aggregation of clutches. In each site we also assessed the presence/absence of the invasive crayfish and the relative abundance in the breeding period. We detected egg clutches in all sites; the crayfish occurred in eight ponds. Our results showed substantial differences between the spawning microhabitat features of the two brown frogs: Rana latastei clutches showed a higher degree of aggregation and were associated with deeper areas of the ponds , while Rana dalmatina deposited more spaced out clutches in areas of the ponds that were less deep. For both species, spawning microhabitat features were not significantly different between sites with and without P. clarkii. Although we did not detect behavioural responses to P. clarkii in the choice of spawning microhabitat , additional studies are required to assess whether these frogs modulate other behavioural traits (e.g. during larval development) in response to the invasive predator.

New pathologies are causing dramatic declines and extinctions of multiple amphibian species. In 2013, in one fire salamander population of Northern Italy, we found individuals with undescribed cysts at the throat level, a malady whose existence has not previously been reported in amphibians. With the aim of describing this novel disease, we performed repeated field surveys to assess the frequency of affected salamanders from 2014 to 2020, and integrated morphological, histological, and molecular analyses to identify the pathogen. The novel disease affected up to 22% of salamanders of the study population and started spreading to nearby populations. Cysts are formed by mucus surrounding protist-like cells about 30 µm long, characterized by numerous cilia/undulipodia. Morphological and genetic analyses did not yield a clear match with described organisms. The existence of this pathogen calls for the implementation of biosecurity protocols and more studies on the dynamics of transmission and the impact on wild populations.

Abstract Accurate measures of species abundance are essential to identify conservation strategies. N-mixture models are increasingly used to estimate abundance on the basis of species counts. In this study we tested whether abundance estimates obtained using N-mixture models provide consistent results with more traditional approaches requiring capture (capture-mark recapture and removal sampling). We focused on endemic, threatened species of amphibians and reptiles in Italy, for which accurate abundance data are needed for conservation assessments: the Lanza’s Alpine salamander Salamandra lanzai, the Ambrosi’s cave salamander Hydromantes ambrosii and the Aeolian wall lizard Podarcis raffonei. In visual counts, detection probability was variable among species, ranging between 0.14 (Alpine salamanders) and 0.60 (cave salamanders). For all the species, abundance estimates obtained using N-mixture models showed limited differences with the ones obtained through capture-mark-recapture or removal sampling. The match was particularly accurate for cave salamanders in sites with limited abundance and for lizards, nevertheless non-incorporating heterogeneity of detection probability increased bias. N-mixture models provide reliable abundance estimates that are comparable with the ones of more traditional approaches, and offer additional advantages such as a smaller sampling effort and no need of manipulating individuals, which in turn reduces the risk of harming animals and spreading diseases.

Abstract Species can show strong variation of local abundance across their ranges. Recent analyses suggested that variation in abundance can be related to environmental suitability, as the highest abundances are often observed in populations living in the most suitable areas. However, there is limited information on the mechanisms through which variation in environmental suitability determines abundance. We analysed populations of the microendemic salamander Hydromantes flavus, and tested several hypotheses on potential relationships linking environmental suitability to population parameters. For multiple populations across the whole species range, we assessed suitability using species distribution models, and measured density, activity level, food intake and body condition index. In high-suitability sites, the density of salamanders was up to 30-times higher than in the least suitable ones. Variation in activity levels and population performance can explain such variation of abundance. In high-suitability sites, salamanders were active close to the surface, and showed a low frequency of empty stomachs. Furthermore, when taking into account seasonal variation, body condition was better in the most suitable sites. Our results show that the strong relationship between environmental suitability and population abundance can be mediated by the variation of parameters strongly linked to individual performance and fitness.

Leeches can parasitize many vertebrate taxa. In amphibians, leech parasitism often has potential detrimental effects including population decline. Most of studies on the host-parasite interactions involving leeches and amphibians focus on freshwater environments, while they are very scarce for terrestrial amphibians. In this work, we studied the relationship between the leech Batracobdella algira and the European terrestrial salamanders of the genus Hydromantes, identifying environmental features related to the presence of the leeches and their possible effects on the hosts. We performed observation throughout Sardinia (Italy), covering the distribution area of all Hydromantes species endemic to this island. From September 2015 to May 2017, we conducted >150 surveys in 26 underground environments, collecting data on 2629 salamanders and 131 leeches. Water hardness was the only environmental feature correlated with the presence of B. algira, linking this leech to active karstic systems. Leeches were more frequently parasitizing salamanders with large body size. Body Condition Index was not significantly different between parasitized and non-parasitized salamanders. Our study shows the importance of abiotic environmental features for host-parasite interactions, and poses new questions on complex interspecific interactions between this ectoparasite and amphibians. Keywords: Parasitism, Cave, Interaction, Leech, Speleomantes, BCI

Most of the world’s mountain glaciers have been retreating for more than a century in response to climate change. Glacier retreat is evident on all continents, and the rate of retreat has accelerated during recent decades. Accurate, spatially explicit information on the position of glacier margins over time is useful for analyzing patterns of glacier retreat and measuring reductions in glacier surface area. This information is also essential for evaluating how mountain ecosystems are evolving due to climate warming and the attendant glacier retreat. Here, we present a non-comprehensive spatially explicit dataset showing multiple positions of glacier fronts since the Little Ice Age (LIA) maxima, including many data from the pre-satellite era. The dataset is based on multiple historical archival records including topographical maps; repeated photographs, paintings, and aerial or satellite images with a supplement of geochronology; and own field data. We provide ESRI shapefiles showing 728 past positions of 94 glacier fronts from all continents, except Antarctica, covering the period between the Little Ice Age maxima and the present. On average, the time series span the past 190 years. From 2 to 46 past positions per glacier are depicted (on average: 7.8).

The fire salamander (Salamandra salamandra) is a relatively common epigean amphibian, widely distributed throughout Europe, which usually gives birth to aquatic larvae. Even if epigean streams represent the most common places in which the species breeds, in some countries caves with underground waters are also used. To improve our understanding of the habitat features allowing successful breeding of salamanders in underground sites, we combined an exhaustive review of the available literature, especially the grey one, with direct observations performed from 2008 to 2017 in several natural and artificial caves of Lombardy, Liguria and Tuscany (Italy), Ariège and Provence (France). We provide a synthesis of published and unpublished caves in which the fire salamander breeding has been observed, along with a synthesis of the investigated ecological, behavioural and morphological traits. The use of underground sites is reported in several published papers and appears to be a common phenomenon not limited to single karst areas. The absence of predators, the relative stability of the aquatic habitats and the possibility to exploit new ecological resources are environmental factors that favour the breeding of the fire salamander. Our synthesis suggests that breeding of fire salamanders in caves is not a random event, but a widespread phenomenon that may be linked to specific biogeographical factors. Further insights may be obtained by performing genetic analyses on both cave and epigean populations, and considering larger landscape scales for ecological studies as well. Gene flow between salamanders that breed in caves and in streams probably occurs, but on the other hand, assortative mating might limit it, thus allowing the conservation of local adaptations driving successful cave colonisation.

Speleomantes are the only plethodontid salamanders present in Europe. Multiple studies have been performed to investigate the trophic niche of the eight Speleomantes species, but none of these studies included hybrid populations. For the first time, we studied the trophic niche of five Speleomantes hybrid populations. Each population was surveyed twice in 2020, and stomach flushing was performed on each captured salamander; stomach flushing is a harmless technique that allows stomach contents to be inspected. We also assessed the potential divergence in size and body condition between natural and introduced hybrids, and their parental species. Previously collected data on Speleomantes were included to increase the robustness of these analyses. In only 33 out of 134 sampled hybrid Speleomantes we recognized 81 items belonging to 11 prey categories. The frequency of empty stomachs was higher in females and individuals from natural hybrid populations, whereas the largest number of prey was consumed by males. We compared the total length and body condition of 685 adult salamanders belonging to three types of hybrids and three parental (sub)species. Three group of salamanders (one hybrid and two parental species) showed significantly larger size, whereas no difference in body condition was observed. This study provided novel ecological information on Speleomantes hybrid populations. We also provided insights into the potential divergence between hybrids and parental species in terms of size and body condition. We discuss our findings, and formulate several hypotheses that should be tested in the future.

Since 2016, a feral population of llama Lama glama has been present in Central Italy after escaping from a zoological garden and starting to reproduce. We updated demographic status and distribution of this population and investigated societal perception towards the llama presence and management in the area through a standard questionnaire. Field data were collected through direct (transects traveled by car and on foot) and indirect (newspapers, social networks and online platforms) research. The feral population appears to be declining. In July 2020, the population was represented by three individuals (one male and two females), identified also through photoidentification, most likely located within a 40-hectare area. The majority of citizens are aware of the presence of feral llamas and show a positive attitude toward them and a negative one toward management actions. The case of feral llamas in Italy is an evident example of unsafe management of a species which should have kept in a zoo and which, once set free, was able to catalyze the attention of the general public. The decline of this population limits the need of drastic management actions that, given the appreciation expressed by people and press toward these animals, would have been at risk of conflict with the public opinion. Removal action should be rapidly taken, i.e., before any demographic rebound and before the population becomes a stable feature of the local landscape.